1. Field of the Invention
This invention relates to flow cytometers, and in particular, relates to a method of standardization of flow cytometers.
2. Description of the Related Art
Standardization of flow cytometers is critical and it is important that instrument performance be reproducible and that results obtained on a particular instrument be comparable from day to day and with results obtained on other instruments.
Previous inventions have utilized a variety of flow cytometer standardization methods. For example, U.S. Pat. No. 5,093,234 discloses a kit containing microbead populations, each of which has the same dye but different fluorescence levels, and on which the microbeads have the same underlying fluorescence characteristics as the sample to be run on the flow cytometer. This kit allows aligning, compensating and/or calibration of a flow cytometer using microbead standards so that data is reproducible from instrument to instrument and from one time to another time on the same instrument but microbead standards do not approach the accuracy of cell standards. The disclosure of this patent and all other patents and patent applications referred to herein is hereby incorporated herein by reference.
U.S. Pat. No. 5,073,497 discloses microbead reference standards, called QC3.TM. beads, for adjustment of a flow cytometer for subsequent measurement of a selected cell or particle sample labeled with two or more dyes. The microbeads are labeled with the same dyes and have substantially the same size which is about the size of the sample. These standards allow adjustment of a flow cytometer for analysis of selected samples which may have multiple fluorescence labels.
In co-pending patent application U.S. Ser. No. 07/671,198, an automated system, QUICK-CAL.RTM., is provided for calibrating and determining performance of a flow cytometer. In this method, statistical data is generated, including the fluorescence threshold and the MESF-slope of the regression line of the plot of histogram channel vs. MESF of standard microbead populations as a function of peak channel number. MESF units are measures of fluorescence intensity expressed as molecules of equivalent soluble fluorochromes. As presented in co-pending U.S. application Ser. No. 07/671,198, the following formula expresses the relationship between MESF value and the slope of the plot of histogram channel vs. MESF as follows: MESF=exp((channel+a constant)/slope). The values obtained are used to show instrument stability. A CLEANSWEEP.TM. gating function sets a numerical threshold and eliminates populations and events which fall below that level. This invention allows rapid, automatic determination of the slopes, fluorescence thresholds (noise levels), linearities, and Target Channels.
The noise level of a flow cytometer, which is the sum of the optical and electronic noise of the instrument, is important in instrument performance. This noise level varies between flow cytometers. On a particular flow cytometer, the noise level varies with changing ambient conditions, with the type of sample being analyzed, with electrical supply and with stability of flow cytometer fluidics. It is an object of this invention to provide a method which allows flow cytometers to be compared over time and with other flow cytometers regardless of noise level. It is another object of this invention to provide a method for setting up a flow cytometer for determination and qualitative evaluation of the noise level of a flow cytometer.
Prior methods of setting and standardizing compensation of flow cytometers usually place unstained cells in the corner of what is discussed herein as the Window of Analysis, resulting in non-uniform compensation across the intensity ranges being used. It is an object of this invention to provide a method resulting in uniform compensation at any intensity range used.
Non-fluorescent microbeads or "blank" microbeads are used to determine fluorescence threshold of a flow cytometer. The blank microbeads are run on the flow cytometer at the same flow cytometer setting where a selected auto fluorescent sample has observable auto fluorescence, and the peak channel position is used as a fluorescence threshold (U.S. Pat. No. 5,089,416).
Utilizing blank microbeads to set up flow cytometers, it has been found by the inventor herein that with a substantial percentage of flow cytometers with compensation circuits as originally set up by the manufacturers, there is a difference in the relative fluorescence of the blank microbeads and unstained cells depending on whether the compensation circuits are turned on or off. When the compensation circuits are not set up correctly, blank microbeads have a lower fluorescence than unstained cells when the compensation circuits are off, but a higher fluorescence than unstained cells when the compensation circuits are on. Flow cytometer operators thus risk losing data on low fluorescence samples on these flow cytometers. It is therefore an object of this invention to provide a method of setting up a flow cytometer which allows determination of whether the compensation circuits have been set up correctly.
It is a further object of this invention to provide a method for setting up a flow cytometer which allows fluorescence compensation to be set and to be uniform across the range of fluorescence intensity used.
It is a further object of this invention a method for setting up a flow cytometer to provide verification of sample preparation and reagent stability.
It is a further object of this invention a method for setting up a flow cytometer to ensure comparability of instrument performance from day to day and from instrument to instrument.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.